This proposal will investigate neural circuits driving negative phototaxis in an emerging model for neural circuit analysis: larvae of the primitive chordate Ciona. Ciona larvae have a number of features that make them ideally suited for this project. They are small and transparent, and have only 177 CNS neurons. Moreover, putative circuits for phototaxis have been identified from the Ciona connectome. Negative phototaxis in Ciona larvae consists of two phases. First, the larvae perform short orienting swims in which they attempt to discern the direction of ambient lighting by moving their bodies. Second, if the larva detects a change in light falling on their photoreceptors as they turn away from the light source, a sustained negative phototactic swim results. However, many orienting swims terminate without a sustained swim. This proposal will examine two aspects of phototaxis: 1) how neural circuits regulate the frequency of orienting swims; and 2) how orienting and sustained swims are linked at the circuit level. Ciona larvae show a wide distribution in the time intervals between short orienting swims. However, analysis of a large dataset of swim interval times points to underlying oscillations governing spontaneous swims frequency, with the dominant period being once every two seconds (O.5 Hz). In preliminary studies we have identified a VGAT-positive neuron that oscillates with a frequency of 0.5 Hz in a region of Ciona CNS called the anterior brain vesicle. We have named the oscillating neuron the anterior brain vesicle oscillator (aBVO). Moreover, mutant and pharmacological studies both point to the aBVO as a likely regulator of spontaneous swim frequency. Proposed experiments in Specific Aim 1 will target the aBVO neuron using optogenetic tools and laser ablation, and then assess the behavioral outcomes. Specific Aim 2 will address the second question: what is the circuit link between short spontaneous orienting and sustained phototactic swims? Our preliminary studies recording GCaMP activity in VACHT-positive neurons suggest a plausible and testable circuit model. We observed that short spontaneous tail movements in larvae were accompanied by Ca2+ transients in the same VACHT-positive interneurons that are the primary targets of the photoreceptors - a neuron class called photoreceptor relay neurons (prRNs). Moreover, the connectome allowed us to identify likely candidate neurons corresponding to the aBVO, based on their 3D locations and connectivities, and the major synaptic targets of these candidate neurons are also the prRNs. Thus, we hypothesize that short orienting swims are initiated in the same circuit as the sustained phototactic swims, and it is the presence or absence of input from the photoreceptors that determines if a sustained swim is initiated. Experiments in Specific Aim 2 will test this hypothesis by targeting the prRNs to determine if they are necessary for the initiation of orienting swims. We also hypothesize that in the absence of aBVO inhibition the prRNs would oscillate. We will test this hypothesis by examining a mutant line that lacks the aBV region of the CNS.

The workings of the human brain at the level of individual nerve cells is poorly understood. Because of the vast complexity of the human brain, researchers have turned to simpler animals. This project will use a simple animal with only 177 nerve cells to investigate how the nervous system is able to use light cues from the environment to navigate.